Supplementary Figure 1

ROC curve for CTC Summation Method

100% - Specificity%

Sen

siti

vity

%

0 50 1000

50

100

150

AUC-ROC= 0.9507 (95% CI: 0.9012 to 1.000)P<0.0001

Supplementary Figure 2

All patients

Characteristic N %

Median Age of patients

58 yrs

ER

Positive 40 69

Negative 18 31

PR

Positive 29 50

Negative 29 50

HER2

Positive 7 12

Negative 51 88

Status

Stable 18 31

Progression 40 69

Supplementary Table 1: Classification of patients based on receptor and disease status

Assay Blood volume per test CTC enrichment principle Detection method

CellSearch Method 7.5 mL Immunomagnetic using EpCAMVisual confirmation of CK+/DAPI+/CD45- cells using automated fluorescence imaging

CSV Method7.5 mL (for comparative analysis)

Depletion of CD45+ cells and positive selection of cell-surface vimentin positive cancer cells using 84-1 antibody

Visual confirmation of CSV+/Cancer specific marker using Confocal Imaging

Supplementary Table 2 Comparison between two detection techniques

Abbreviations: CSV= Cell-surface vimentin, CK= Cytokeratin,

K coefficient Degree of Agreement Agreement (%)

Comparison of Progressive population with the CellSearch CTC test

-0.017 Poor 45%

Comparison of Stable population with the CellSearch CTC test

0.440 Moderate 66.67%

Supplementary Table 3 Concordances between CSV and CellSearch detection techniques

Abbreviations: CSV= Cell-surface vimentin, CTC= Circulating tumor cells.

  Triple Negative Luminal A Luminal B HER2 Type

CTCs<5  Stable Progression Stable Progression Stable Progression Stable Progression

CSV < 5 4 4 10 2 2 0 1 0

CellSearch < 5 2 8 10 12 2 1 1 0

CTCs≥ 5

CSV ≥ 5 0 8 1 22 0 3 0 1

CellSearch ≥ 5 2 4 1 12 0 1 0 1

CSV: Cell-surface vimentin; CTCs: Circulating tumor cells; ER: Estrogen receptor; PR: Progesterone receptor ; HER2: HER2/neu receptor; Triple negative: ER- PR- HER2-; Luminal A: ER/PR+ HER2- Luminal B: ER/PR+ HER2+, HER2 Type: ER/PR- HER2+

Supplementary Table 4: Receptor based classification for individual samples with stable/progression status as determined by both techniques

Triple Negative (n=16)

Sensitivity SpecificityPositive Predictive

ValueNegative Predictive

Value

CSV 66.67% 100% 100% 50%

CellSearch 33.34% 50% 66.67% 20%

Summation (Th 5) 83.33% 25% 76.92% 33.33%

Summation (Th 8) 83.33% 75% 90.91% 60%

Luminal A (n=35)

CSV 91.67% 90.91% 95.65% 83.33%

CellSearch 50% 90.91% 92.3% 45.45%

Summation (Th 5) 95.83% 72.73% 88.46% 88.89%

Summation (Th 8) 95.83% 81.82% 92% 90%

Supplementary Table 5: Analysis of Sensitivity, Specificity, Positive Predictive Value, and Negative Predictive Value based on receptor status of patients

CSV: Cell-surface vimentin Summation: sum of CTC counts from 84-1 and CellSearch methods. Triple negative: ER- PR- HER2-; Luminal A: ER/PR+ HER2- Th 5 indicates a threshold of 5 CTCs/ 7.5 mL as cutoff.Th 8 indicates a threshold of 8 CTCs/ 7.5 mL as cutoff.

n=20 Progression Stable

Nuclear EpCAM 13 1

No Nuclear EpCAM 2 4

Supplementary Table 6: Analysis of Nuclear EpCAM Status

Sensitivity Specificity Positive Predictive Value Negative Predictive Value

n=20 86.67% 80% 92.86% 66.67%

Supplementary Table 7: Sensitivity, Specificity, Positive prediction and Negative prediction values for Nuclear EpCAM method

Sample Age ER/PR/HER2 status Treatment Disease status 84-1 CellSearchS1 65 ER+/PR-/HER2+ Taxol Progression 20 1S3 42 ER+/PR+/HER2- letrozole Stable 0 0S4 48 ER+/PR+/HER2+ trastuzumab Stable 0 0S5 61 ER+/PR+/HER2- paclitaxel Stable 1 0

S7 78 ER+/PR+/HER2- everolimus, exemestane and zoledronic acid Progression 1 2S9 53 ER+/PR+/HER2+ Ixabepilone plus capecitabine, zoladex + AI Progression 32 74S10 44 ER-/PR-/HER2+ docetaxel, pertuzumab and trastuzumab Progression 12 44S11 27 ER-/PR-/HER2- Abraxane and bevacizumab Progression 2 22S12 72 ER+/PR+/HER2- tamoxifen Progression 0 10S13 58 ER+/PR+/HER2- Taxol Progression 5 69S14 67 ER+/PR+/HER2- letrozole Stable 0 0S15 61 ER-/PR-/HER2- Taxol and Avastin Stable 2 1S16 49 ER+/PR+/HER2- Afinitor and tamoxifen Progression 7 54S17 46 ER-/PR-/HER2- eribulin Progression 73 4S18 37 ER-/PR-/HER2- Taxol Stable 1 4

S19 69 ER+/PR-/HER2- everolimus, exemestane and zoledronic acid Stable 2 1S20 64 ER+/PR+/HER2- Taxol Stable 0 1S22 31 ER+/PR+/HER2+ Taxol Stable 3 4S23 53 ER+/PR-/HER2- capecitabine Progression 6 13S24 66 ER+/PR+/HER2- docetaxel and zoledronic Progression 8 11S26 58 ER-/PR-/HER2- eribulin Progression 1 3S27 48 ER-/PR-/HER2- gemcitabine and carboplatin Progression 17 1S28 36 ER+/PR+/HER2- Gemzar, carboplatin Progression 24 1S29 47 ER-/PR-/HER2- MK-2206 Stable 4 11S30 52 ER+/PR+/HER2- tamoxifen Stable 4 1S31 40 ER-/PR-/HER2- Taxol and Avastin Progression 48 1S33 42 ER+/PR-/HER2- abraxane plus bevacizumab Progression 6 34S35 67 ER+/PR+/HER2- everolimus Progression 8 1

S36 68 ER+/PR+/HER2- everolimus, exemestane and zoledronic acid Stable 0 9S38 51 ER+/PR+/HER2- Avastin and eribulin Progression 13 3S39 65 ER-/PR-/HER2- Taxol and Avastin Stable 0 5

S40 60 ER+/PR-/HER2- everolimus, exemestane and zoledronic acid Progression 15 1S41 57 ER-/PR-/HER2- Gemzar Progression 23 74S42 62 ER+/PR-/HER2- vinorelbine Progression 16 7S43 54 ER+/PR-/HER2+ Tamoxifen Progression 66 221S44 41 ER+/PR+/HER2- Abraxane Progression 6 3S45 56 ER-/PR-/HER2+ Pertuzumab, trastuzumab, docetaxel Stable 1 1S46 35 ER-/PR-/HER2- erlotinib plus pralatrexate Progression 60 1S47 69 ER+/PR-/HER2- everolimus and exemestane Progression 30 10S49 66 ER-/PR-/HER2- cisplatin and etoposide Progression 1 55S50 67 ER+/PR+/HER2- Arimidex Stable 0 0S52 67 ER+/PR+/HER2- letrozole Stable 9 0S53 65 ER+/PR-/HER2- anastrozole Progression 7 2S54 52 ER+/PR+/HER2- everolimus and exemestane Stable 0 0S55 63 ER-/PR-/HER2- capecitabine Progression 2 0S56 47 ER-/PR-/HER2- Avastin and eribulin Progression 148 369S57 59 ER+/PR+/HER2- Taxol and Avastin Progression 24 3S58 66 ER-/PR-/HER2- Abraxane Progression 96 2S59 41 ER+/PR+/HER2- eribulin Progression 6 2S60 59 ER+/PR+/HER2- Avastin and eribulin Progression 6 60S61 92 ER+/PR+/HER2- fluoxymesterone Progression 28 0S62 45 ER+/PR+/HER2- gemcitabine Progression 142 26S63 76 ER+/PR-/HER2- fulvestrant Stable 0 0S64 59 ER+/PR+/HER2- anastrozole Progression 6 21S65 67 ER+/PR+/HER2- everolimus and exemestane Progression 180 0S66 30 ER-/PR-/HER2- T-FAC Progression 12 0S67 58 ER+/PR-/HER2- Abraxane and Avastin Progression 30 124S68 40 ER+/PR+/HER2- exemestane, everolimus, and denosumab Progression 144 2

Supplementary Table 8: Clinical parameters, disease status and CTC counts from patients